FR3049060A1 - DEVICE FOR PREPARING A BLOOD SAMPLE - Google Patents

Abstract

The invention relates to a device for preparing a blood sample, characterized in that it comprises a micro-fluidic card (1) which comprises: A first chamber (11) into which said injection channel opens, said first chamber being dedicated to carrying out an extraction operation of proteins to be analyzed present in said blood sample, a second chamber (12) connected to said first chamber so as to receive a first sample comprising the analyte proteins extracted in said first chamber, said second chamber being dedicated to carrying out an operation for digesting the proteins to be analyzed, A third chamber (13) connected to said second chamber so as to receive a second sample including the digested peptides and the non-proteins digested, said third chamber being dedicated to the implementation of a purification and stabilization operation n digested peptides.

Description

Device for preparing a blood sample

Technical field of the invention

The present invention relates to a device for preparing a blood sample and to a system employing said device for preparing a blood sample. The invention also relates to a method for preparing a blood sample implemented with said device.

STATE OF THE ART Proteomic analysis based on mass spectrometry for detecting and assaying proteins in biological samples such as plasma is more and more widespread because it is particularly efficient. However, for this analysis to be optimal and reliable, it requires that the blood sample analyzed be perfectly stable. However, it turns out for example that the time elapsing between the collection of a blood sample, its preparation (for obtaining the serum or plasma fraction) and its transfer to an analysis laboratory is very variable, thus giving analytical results of various qualities. Indeed, the natural degradation process of the proteins of interest present in the plasma can be more or less advanced depending on the time that elapses between the collection of the sample and its transfer to the analysis laboratory, resulting in some variability in the results obtained.

The object of the invention is to propose a device which is used to prepare and stabilize a blood sample, this device making it possible to effectively treat a blood sample by reducing the time that elapses between the blood sample and the stabilization of the sample , so as to obtain good quality results that are perfectly reproducible.

Presentation of the invention

This object is achieved by a device for preparing a blood sample, comprising a microfluidic card which comprises: an injection channel through which said blood sample can be injected,

A first chamber into which said injection channel opens, said first chamber being dedicated to carrying out an extraction operation of proteins to be analyzed present in said blood sample,

A second chamber connected to said first chamber so as to receive a first sample comprising the proteins to be analyzed extracted in said first chamber, said second chamber being dedicated to carrying out an operation of digestion of the proteins to be analyzed present in order to obtain a second sample including digested peptides and undigested proteins,

A third chamber connected to said second chamber so as to receive said second sample including digested peptides and undigested proteins, said third chamber being dedicated to carrying out an operation for purifying and stabilizing the digested peptides.

According to a first configuration, the device comprises a depletion support present in the first chamber for capturing majority proteins present in said blood sample.

In this first configuration, three alternative embodiments can be envisaged, taken alone or in combination: according to a first variant embodiment, the depletion support comprises beads present in the first chamber and grafted with one or more capture components adapted to majority protein to capture. According to a second variant embodiment, the depletion support comprises pillars positioned in the first chamber and grafted with one or more capture components adapted to the majority proteins to be captured. According to a third variant embodiment, the first chamber is in the form of a coil whose functionalized surfaces form the depletion support.

According to a second configuration, the device comprises an enrichment support present in the first chamber for fixing and enriching the proteins to be analyzed present in the blood sample.

Similarly, in this second configuration, several variants can be envisaged, taken alone or in combination: according to a first variant embodiment, the enrichment support comprises beads present in the first chamber and grafted with one or more components of enrichment adapted to the proteins to be enriched. According to a second variant embodiment, the enrichment support comprises pillars positioned in the first chamber and grafted with one or more enrichment components adapted to the proteins to be enriched. According to a third variant embodiment, the first chamber is in the form of a coil whose surfaces form the enrichment support.

In the second chamber, several solutions can be envisaged for the digestion operation. These solutions are the following, taken alone or in combination:

The device comprises a channel opening into the second chamber, for an injection of an enzyme or a mixture of enzymes for carrying out the digestion operation.

The second chamber comprises a surface at least partially covered with an enzyme or a mixture of enzymes for carrying out the digestion operation.

The second chamber has an internal volume for storing an enzyme or a mixture of enzymes in lyophilized form for carrying out the digestion operation.

According to another feature, the device comprises separation means arranged to separate the plasma or serum blood cells present in the blood sample.

According to another feature, the device comprises a reverse phase liquid chromatography column housed in the third chamber for hooking, stabilizing and / or separating the digested peptides.

According to another feature, the device comprises:

A first valve arranged on the injection channel for controlling an opening or closing of said channel, a second valve arranged to control the transfer of the first sample from the first chamber to the second chamber,

A third valve arranged to control the transfer of the second sample from the second chamber to the third chamber. The invention relates to a system for preparing a blood sample comprising a programmable automaton and a device for preparing a blood sample as defined above. The programmable logic controller comprises a processing unit intended to execute software modules adapted to generate commands for actuators in a sequence determined according to the different steps of preparation of the blood sample on said device. The invention also relates to a method for preparing a blood sample, implemented in the device as defined above, the method comprising the following steps:

Extraction in the first chamber of proteins to be analyzed present in said blood sample,

Digestion in the second chamber of the proteins to be analyzed present in a first sample comprising the proteins to be analyzed extracted in said first chamber in order to obtain a second sample including digested peptides and undigested proteins, - Separation in the third chamber between the digested peptides and undigested proteins.

According to one particularity, the extraction step is carried out by depletion of the majority proteins or enrichment of the proteins to be analyzed present in the blood sample.

According to another particularity, the separation step is carried out by liquid chromatography on a reverse phase column.

BRIEF DESCRIPTION OF THE FIGURES Other features and advantages will appear in the following detailed description with reference to the accompanying drawings in which:

Figure 1 schematically shows a system for preparing a blood sample, including the device for preparing the blood sample according to the invention.

FIG. 2 represents a diagram showing the different steps of the method for preparing a blood sample, implemented using the device of FIG. 1.

FIGS. 3A and 3B show two chromatograms, resulting from a mass spectrometric analysis performed on proteins respectively derived from a preparation carried out partly on the device of the invention and a preparation carried out manually and in a conventional manner.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The invention relates to a device for preparing a blood sample before its analysis, for example by liquid chromatography or by quantitative mass spectrometry. The device of the invention will make it possible in particular to prepare and store a stabilized sample for subsequent analysis, without risk of degradation of the sample.

In the remainder of the description, in a nonlimiting manner, the device of the invention will be described for a preparation from a plasma sample derived from the blood sample taken from the patient, but it should be understood that it could be used for a preparation from the serum from the sample.

In the remainder of the description, the term "room" means a space defining a non-zero or partially closed closed volume into which one or more channels may open.

The device mainly comprises a micro-fluidic card which includes channels and several chambers each contributing to the preparation of the blood sample before analysis. This device can be used in a more comprehensive system of blood preparation. This system comprises in particular a programmable controller 2 which controls the preparation of the blood sample on the card.

As described above, the device of the invention comprises a microfluidic card 1, having for example the format of a credit card, in which all the steps leading to the preparation and stabilization of the sample before his analysis can be conducted.

The card 1 is for example placed in a suitable housing of the programmable controller. The programmable logic controller 2 comprises a processing unit arranged to control all the steps necessary for the preparation of the blood sample on said card. The processing unit 2 executes software modules each intended to control one or more of these steps. These software modules are executed to generate output commands for different actuators. These actuators may be for example micro-pumps, magnetic stirring systems, heating systems, cooling systems, ultrasonic or microwave emission systems, micro-fluidic valves. These various actuators are for example part of the system of the invention used for the preparation of the blood sample in the card.

This microfluidic card 1 is for example made using two plates bonded to one another and on each of which are formed the patterns forming the chambers and fluidic paths of the card. The card 1 is for example made of a transparent material of the type PMMA, polycarbonate, COC (for "Cylcic Olefin Copolymer").

With reference to FIG. 1, the card mainly comprises: an injection channel 10 through which said blood sample may be injected; a first chamber into which said injection channel opens out; a second chamber 12 situated downstream; of the first chamber, - A third chamber 13 located downstream of the second chamber.

Valves positioned in a suitable manner on the card 1 are controlled by the programmable logic controller 2 to control the different steps of preparation of the blood sample on the card. The device thus comprises for example at least: A first valve V1 positioned on the injection channel to control the injection of the liquid flow formed by the blood sample to be prepared in the first chamber. - A second valve V2 positioned on a channel connecting the first chamber to the second chamber and for controlling the passage of liquid from the first chamber to the second chamber.

A third valve V3 positioned on a channel connecting the second chamber to the third chamber and for controlling the passage of liquid from the second chamber to the third chamber.

One or more other valves V4, V5, V6 each intended to control the injection to one of said chambers of a component necessary for the preparation of the blood sample or to remove residues.

Furthermore, micropumps (not shown) are also controlled by the programmable controller 2 to pass the sample from one chamber to another, by suction or injection. The programmable controller 2 thus comprises a plurality of output modules each intended to control a separate actuator, such as each valve or each micro-pump.

Referring to Figure 1 and Figure 2, the device of the invention and its use for carrying out a method of preparing a blood sample are explained below.

The valves V1, V2, V3, V4, V5, V6 are all initially in the closed state.

More specifically, the injection channel 10 is intended to receive the blood sample taken from the patient. The tube containing the blood sample will for example be connected to the inlet 100 of the injection channel. This inlet 100 may be made according to different embodiments depending on the type of fluidic connection chosen. Different types of connection may indeed be envisaged, employing for example a module type "Saf-T Wing" (registered trademark). The injection of the blood sample into the channel 10 will for example be controlled by the first valve V1 controlled by the automaton and activated by a microprocessor controlled by the automaton. The initial blood sample taken from the patient's arm will be, for example, 2 to 10 milliliters. The analysis will be performed on a few microliters at 50 μΙ of plasma (or serum) contained in this sample.

The injection channel 10 opens into the first chamber 11 formed on the card 1. This first chamber 11 is dedicated to the extraction of the proteins of interest from the plasma of the blood sample. This extraction may be carried out: by depletion of the major proteins present in the plasma, or by affinity enrichment of the targeted plasma proteins.

In the first step E1 defined in FIG. 2, to effect the separation between the plasma and the blood cells in the blood sample, several solutions can be envisaged: a first solution consists in producing it in the first chamber using a polymer, for example a Dacron type polymer (registered trademark) which allows separation by filtration between the plasma and the cells. A second solution consists in using a Vivid (registered trademark) membrane membrane placed in the path of the injection channel 10 or directly on the cartridge which initially comprises the blood sample to be analyzed and which is positioned on the inlet 100. of the canal during the injection of the blood sample.

A third solution is to use a capillary micro-channel 10 into which the blood sample is injected.

The second step E2 defined in FIG. 2 is carried out in the first chamber 11. It consists of depleting the majority proteins present in the plasma sample in order to capture them or enriching the proteins to be analyzed present in this sample.

In the first chamber 11, the operation of depletion of the major proteins present in the plasma is for example carried out using a support allowing the capture of one or more major proteins present in the plasma sample, for example albumin. . As represented in FIG. 1, this support will for example be magnetic beads 110 housed and held in the first chamber 11 or injected via a channel adapted inside the chamber by a command sent by the programmable logic controller 2. The balls Magnetic 110 employees will be grafted with majority protein capture components in order to trap them in the first chamber. It will be for example:

Magnetic beads grafted with Cibacron Blue for the capture of albumin, and / or

Magnetic beads grafted with protein A for immunoglobulin G capture, and / or

Beads grafted with antibodies or ligands for the specific capture of the major proteins of the plasma.

As an alternative embodiment or in addition to the balls 110 used in the first chamber 11, the capture support may comprise pillars positioned in the first chamber 11, the surface of which is coated with one or more of the capture components mentioned above. . The pillars are fixed in the chamber and positioned judiciously so that the plasma sample flows between the pillars and come into contact with them to capture a maximum of the majority proteins.

In addition to or replacement of the balls 110 and / or abutments mentioned above, the surfaces of the chamber may be coated with one of the capture components mentioned above in order to trap the majority proteins. The shape and configuration of the first chamber 11 are then optimized to maximize the exchange surfaces between the plasma sample and the walls that define the internal volume of the chamber. Advantageously, to maximize the exchange surfaces, the first chamber is in the form of a serpentine channel.

It is therefore understood that the various means and arrangements provided on the map and described above for trapping the majority proteins can be combined together in a judicious manner to obtain a better result. For example, it will be a question of using a serpentine channel whose surfaces are coated with one or more components for capturing the majority proteins, this channel opening into the first chamber 11 in which beads and / or abutments are placed. as described above.

In the case where an enrichment operation of the targeted minority proteins is carried out in replacement of a depletion operation of the majority proteins present in the plasma sample, the enrichment support is grafted with specific antibodies. As for the depletion, the support can be constituted and arranged according to one of the variants described above for the depletion operation. After washing, the fraction fixed on the enrichment support used will be eluted towards the second chamber 12.

Once the depletion or enrichment operation is carried out, a plasma sample which contains the proteins of interest is then transferred to the second chamber of the card, by an opening command of the second valve V2 and a control of a micro-pump for sucking or injecting the sample to the second chamber 12. This is then the third step E3 of the preparation of the blood sample which consists of an operation of digestion of the targeted proteins.

The second chamber 12 is dedicated to the digestion of the targeted proteins contained in the plasma sample present. To perform this digestion, the programmable controller 2 controls the valve V5 at the opening and a micro-pump for injecting an enzyme or a mixture of proteolytic enzymes via a specific channel 120 which opens into said chamber 12. The programmable controller then controls a magnetic stirring system and a heating system to heat the chamber to a predetermined temperature to ensure the protein digestion reaction. As an alternative embodiment or in addition, the surface of the second chamber may be coated with the enzyme or enzyme mixture necessary for digestion. In another variant embodiment or in addition, it is possible to imagine that the enzyme or the enzyme mixture is present in the space formed by the second chamber, for example in a freeze-dried form.

Once the digestion process is completed in the second chamber 12, the plasma sample which then contains digested peptides and undigested proteins is transferred to the third chamber 13. The automaton 2 sends an opening command to the third V3 valve and a control micropump to transfer the plasma sample to the third chamber 13. This is then the fourth step E4 of the blood sample preparation process.

The third chamber is dedicated to the attachment of digested peptides for the purpose of separating and purifying them. For this, use is made, for example, of a reverse phase liquid chromatography column, for example of the C4 or C18 type, on which the digested peptides are retained and can be washed and then stabilized. A washing liquid can be injected into the third chamber 13 via a channel 130 formed on the card and controlled by a valve V6. In addition or as an alternative embodiment, it is possible to implement an ultrafiltration operation which makes it possible to retain the undigested proteins and to select only the peptides digested for stabilization. This filter will for example be positioned at the outlet of the second chamber 12. The establishment of a pressure may be necessary to push the sample through this filter.

During this fourth step E4, the digested peptides are stabilized in the map. Stabilization can be achieved by various methods, for example by fixing on resin type C4 or C18, by drying, by freezing ... To stabilize, the PLC 2 sends a command to a cooling system or a system drying by heating or by passage of air, arranged to act on the third chamber to stabilize the peptides present.

Once the peptides are stabilized, they can be stored in the card, in the third chamber 13 or in a specific storage chamber.

For their analysis, for example by mass spectrometry, the peptides which have been stabilized (by drying, freezing or any other suitable method ...) are eluted from the card 1 or are directly loaded onto a resin or a liquid chromatography column. reverse phase of type C4 or C18 present in the third chamber.

The principle of the invention which consists in using the same card to carry out all the steps of preparation of the blood sample has many advantages and makes it possible to obtain particularly convincing results. It has indeed been found that the eluted and analyzable quantity of the different peptides resulting from the digestion of the targeted proteins of interest is much greater when the preparation has been carried out automatically on a single device according to the invention than on the following various steps performed successively manually. Figures 4A and 4B show the quality of the results obtained. The intensity of the chromatography peaks are much greater for the peptides analyzed after stabilization on the device of the invention (FIG. 4A) than for the peptides analyzed after a conventional manual preparation.

Claims (19)

1. Device for preparing a blood sample, characterized in that it comprises a micro-fluidic card (1) which comprises: an injection channel (10) through which said blood sample may be injected, a first chamber ( 11) into which said injection channel opens, said first chamber being dedicated to carrying out an extraction operation of proteins to be analyzed present in said blood sample, A second chamber (12) connected to said first chamber of to receive a first sample comprising the extracted analyte proteins in said first chamber, said second chamber being dedicated to the implementation of an operation of digestion of the present analyte proteins to obtain a second sample including digested peptides and undigested proteins, A third chamber (13) connected to said second chamber to receive said two xth sample including digested peptides and undigested proteins, said third chamber being dedicated to carrying out an operation for purifying and stabilizing the digested peptides. 2. Device according to claim 1, characterized in that it comprises a depletion support present in the first chamber (11) for capturing major proteins present in said blood sample. 3. Device according to claim 2, characterized in that the depletion carrier comprises beads (110) present in the first chamber and grafted with one or more capture components adapted to the majority proteins to be captured. 4. Device according to claim 2 or 3, characterized in that the depletion support comprises pillars positioned in the first chamber (11) and grafted with one or more capture components adapted to the majority proteins to be captured. 5. Device according to one of claims 2 to 4, characterized in that the first chamber (11) is formed in the form of a coil whose functionalized surfaces form the depletion support. 6. Device according to claim 1, characterized in that it comprises an enrichment support present in the first chamber (11) for fixing and enriching the proteins to be analyzed present in the blood sample. 7. Device according to claim 6, characterized in that the enrichment support comprises beads present in the first chamber and grafted with one or more enrichment components adapted to the proteins to be enriched. 8. Device according to claim 6 or 7, characterized in that the enrichment support comprises pillars positioned in the first chamber and grafted with one or more enrichment components adapted to the proteins to be enriched. 9. Device according to one of claims 6 to 8, characterized in that the first chamber (11) is formed in the form of a coil whose surfaces form the enrichment support. 10. Device according to one of claims 1 to 9, characterized in that it comprises a channel (120) opening into the second chamber (12) for an injection of an enzyme or a mixture of enzymes for the implementation of the digestion operation. 11. Device according to one of claims 1 to 9, characterized in that the second chamber comprises a surface at least partially covered with an enzyme or a mixture of enzymes for carrying out the digestion operation. . 12. Device according to one of claims 1 to 9, characterized in that the second chamber comprises an internal volume for storing an enzyme or a mixture of enzymes in freeze-dried form for the implementation of the digestion operation. 13. Device according to one of claims 1 to 12, characterized in that it comprises separation means arranged to separate the plasma and blood cells present in the blood sample. 14. Device according to one of claims 1 to 13, characterized in that it comprises a reverse phase liquid chromatography column housed in the third chamber to hang, stabilize and / or separate the digested peptides. 15. Device according to one of claims 1 to 14, characterized in that it comprises: - a first valve (V1) arranged on the injection channel for controlling an opening or closing of said channel, A second valve (V2 ) arranged to control the transfer of the first sample from the first chamber to the second chamber, A third valve (V3) arranged to control the transfer of the second sample from the second chamber to the third chamber. 16. System for preparing a blood sample comprising a programmable automaton, characterized in that it comprises a device for preparing a blood sample according to one of claims 1 to 15 and in that the programmable logic controller comprises a processing unit for executing software modules adapted to generate commands to actuators in a sequence determined according to the different steps of preparing the blood sample on said device. 17. A method for preparing a blood sample, characterized in that it is implemented in the device as defined in one of claims 1 to 15 and in that it comprises the following steps: Extraction in the first chamber of proteins to be analyzed present in said blood sample, digestion in the second chamber of the proteins to be analyzed present in a first sample comprising the proteins to be analyzed extracted in said first chamber in order to obtain a second sample including digested peptides and undigested proteins, - Separation in the third chamber between the digested peptides and the undigested proteins. 18. The method of claim 17, characterized in that the extraction step is carried out by depletion of the major plasma proteins or enrichment of the proteins to be analyzed. 19. The method of claim 17 or 18, characterized in that the separation step is carried out by liquid chromatography on a reverse phase column type.